“Bone Marrow on X chromosome” kinase (BMX, also termed ETK) is a non-receptor tyrosine kinase and is activated downstream of phosphatidylinositol-3 kinase (PI-3K) and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC), but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphotyrosine (pY) in the −1 position. Potential substrates include multiple tyrosine kinases with kinase domain pYpY sites required for full activity. BMX has been found to phosphorylate residue Y577 of focal adhesion kinase (FAK) subsequent to Y576 phosphorylation by SRC. In addition, BMX loss by RNA interference and mouse embryonic fibroblasts (MEFs) from Bmx negative (Bmx−) mice displayed impaired FAK signaling. Insulin receptor (IR) phosphorylation similarly was decreased by BMX loss, as was hepatic IR phosphorylation in Bmx− mice. However, glucose tolerance was increased, reflecting a marked compensatory decrease in the activity of the AKT phosphatase PHLPP. These findings reveal a mechanism through which BMX functions as a central regulator of multiple kinase pathways.
Tec kinases, which include TEC, BTK, ITK, RLK/TXK, and BMX, are non-receptor tyrosine kinases expressed primarily in lymphoid and myeloid lineages. They are related in structure to SRC in that they have an SH3 domain followed by an SH2 domain and tyrosine kinase domain but lack the C-terminal tyrosine that negatively regulates SRC kinases (Afar et al., Mol. Cell. Biol. 16, 3465 (1996); Andreotti et al., Nature 385, 93 (1997); Nore et al., Biochim. Biophys. Acta 1645, 123 (2003); Park et al., Immunity 4, 515 (1996)). (FIG. 1A). The Tec kinases are unique in having a pleckstrin homology (PH) domain that mediates membrane targeting in response to PI-3K activation by binding to phosphatidylinositol 3,4,5-triphosphate (PIP3) (Qiu et al., Oncogene 19, 5651 (2000)), which results in SRC-mediated phosphorylation of a kinase domain tyrosine that activates the enzyme. Mutations in BTK, which is restricted to B cells, cause X-linked agammaglobulinemia (de Weers et al., Hum. Mol. Genet. 3, 161 (1994)), while loss of ITK expressed in T cells results in a variety of T cell defects (Andreotti et al., Cold Spring Harb. Perspect. Biol. 2, a002287 (2010)). BMX is broadly expressed by cell types outside the lymphoid/myeloid lineage including arterial endothelium and epithelial cells (Chott et al., Am. J. Pathol. 155, 1271 (1999); Rajantie et al., Mol. Cell Biol. 21, 4647 (2001); Robinson et al., Proc. Natl. Acad. Sci. U.S.A. 93, 5958 (1996); Tamagnone et al., Oncogene 9, 3683 (1994)). While Bmx− mice have only a modest defect in ischemia-induced angiogenesis (Rajantie et al., Mol. Cell Biol. 21, 4647 (2001); He et al., J. Clin. Invest. 116, 2344 (2006); Zhang et al., J. Biol. Chem. 278, 51267 (2003); Pan et al., Mol. Cell Biol. 22, 7512 (2002)), increasing evidence indicates that BMX has diverse modulatory roles in multiple cellular processes (Tu et al., Cancer Res. 68, 2861 (2008); Jiang et al., J. Biol. Chem. 279, 50181 (2004); Kim et al., J. Biol. Chem. 277, 30066 (2002); Bagheri-Yarmand et al., J. Biol. Chem. 276, 29403 (2001); Chau et al., Oncogene 21, 8817 (2002); Semaan et al., J. Immunol. 180, 3485 (2008)). However, the direct downstream targets of BMX remain elusive, and substrate motifs for BMX and other Tec kinases have not been identified.